The answers comprise a large body of knowledge graced in several ways. First,
histology is colourful. Secondly, almost everything seen is actually there;
which is not to say that what is not seen is absent. Third, one handles and
views actual slides - the source material for most of histology, not just
someone else's selected images. Fourth, the structures can be interpreted
as parts in developmental and functional sequences, and be fitted together
by satisfying accounts, for example, of how cells defend the body. So much is now known of the roles of cells and
structures that histology is both descriptive microanatomy, and an
introduction to function for the whole body. Powerpoint
Dead Living (a)Section - a thin slice of Such preparations may be out of the tissue or organ - on a glass slide body in a tissue culture system, or or metal grid. living within the body but in an (b)Smear on a glass slide - observable situation, e.g., a suitable for suspensions, e.g., transparent chamber inserted into blood, urine, mucus, cyst fluid, the ear or skin. The first need is bone marrow, etc. to keep the preparation alive. This (c)Spread sheet of tissue seriously limits the facilities for stretched thin, e.g., areolar observation. For example, staining connective tissue. is usually impracticable. Thus, (d)Teased apart fibrous phase-contrast or interference-contrast elements, e.g., muscle. microscopy has to be used in order to overcome the poor contrast between natural structures.
2 Steps needed to make and study a histological section
The only way to improve resolving power is to reduce substantially the
wavelength of the light. This is achieved by the electromagnetic beam of the
electron microscope. The beam is focused through the object suspended on its
metal grid, and is magnified before striking a fluorescent screen to be
transformed into a visible image (Chapter 30.K). [Caution!
the link takes you there , but 'back' brings you only to the start of the last
Chapter that you linked to.]
The resolutions so far achieved in biology with transmission electron microscopy (TEM/EM) are of the order of 1 nm at a magnification of X 2 000 000. The other forms of microscopy - phase-contrast, interference, fluorescence, confocal scanning, atomic-force (and X-ray diffraction) - will be discussed in Chapter 30. in relation to the problems for which they are suited.
2 Microscopy for the student (may not apply in toto to the reader's use)
(c) Before plugging in the microscope, check to feel how the switch and
rheostat work. Plug in, switch on, and adjust the rheostat up one third of its
range to start.
(d) Otherwise, use artificial light provided by an electric bulb behind a ground-glass screen to furnish a constant and reliable source. Light intensity can be increased by bringing the lamp nearer to the mirror, if the lamp is not built-in.
(e) If the condenser in use (nearly always), use the plane side of the mirror, if the lamp is not built-in.
(f) Raise the condenser to very near the underside of the stage, and open the iris diaphragm.
(g) Place a clean, stained slide on the stage and using the coarse and fine focusing controls bring it into focus with X l0 objective.
(h) With the condenser racking knob focus the light source on the specimen. This has happened when the specimen itself is in focus and some aspects of the light source is also seen sharply defined, e.g., the bulb filament or scratches on the frosted glass screen. If this feature of the light source is obtrusive, now place the condenser very slightly out of focus. Do not lower the condenser way out of focus as a means to reduce the light intensity.
(i) The iris diaphragm should now be closed just to the point where glare is eliminated. Further closure will make the field too dark and reduce resolving power.
(j) The microscope is now set up for use, but the requirements change for each objective. Higher power objectives require more light thus the iris will need to be opened and perhaps the lamp brought nearer to the mirror and the condenser refocused.
(k) Note that the objective lenses are of different lengths, and they are not always parfocal. Be careful when switching in a higher power lens that it does not hit the slide because of its greater length. Clean the lenses only with lens paper.
(l) If the X 44 objective will not focus to a clear image, check first that the slide is not upside down on the stage.
(m) Use of the 'oil immersion' lens:
... (i) Select field of interest with the high dry lens (X40); centre precisely the cell or object in the microscopic field; if X 95 lens is already mounted go to (v).
... (ii) Raise the objective lens assembly and remove the low power (X4) lens.
... (iii) Place it in the container to be found on the door of the microscope cabinet from which you have taken the oil immersion (X 95) lens.
... (iv) Screw the oil lens into the now vacant place on the objective nosepiece.
... (v) Place carefully one drop of immersion oil from the small bottle issued on the area of the slide to be studied.
... (vi) Switch round the objective nosepiece to bring the oil immersion lens into play.
... (vii) Very carefully lower the objective assembly with the coarse focusing, until the tip of the oil lens touches the drop of oil. This operation must be controlled by observing the descending lens from the side. Do not yet look down through the eyepiece. Once the lens has touched the oil raise it slightly, but not so far that the drop breaks away.
... (viii) Look in the eyepiece and focus down with the fine focusing control very slowly and gently until the specimen comes into focus. If you seem to have gone down a very long way without a clear image, again check from the side that you have not overshot and the lens is not nearly on the glass of the coverslip. If this has happened raise the lens slowly, while looking for a focused image.
... (ix) The oil objective lens needs much light so that the iris diaphragm may have to be opened.
... (x) As soon as you have finished using the oil lens, raise (remove) and clean it. (Replace X 4 lens on the nosepiece and the oil lens in its box.) Clean the slide of oil with lens or tissue paper. Do not allow oil to get on to the other, dry, lenses.
(n) Other controls the class microscope may have include eyepiece focusing,
filter-holders, centring screws for the condenser, and a rheostat, lens and
light-stop for the light source. Ask for instructions in their use and for
help with any mechanical problem.
(o) Take care of the microscope, carry it only by its arm, protect it from dust by keeping it locked in its case, and do not stand it or boxes of slides near the edge of the bench. If lens paper alone is insufficient to clean a lens, use no solvents but consult a demonstrator. On no account exchange the lenses of your microscope for those of any other microscope.
Spectacle-wearers need not use their glasses in microscopy; if they do, they should beware of damaging their glasses, while trying to compensate for the narrowed field of view.
3 Differences between light and electron microscopy
l Chapters 2 and 3 deal with microscopic details of cells - cytology, for which EM is better suited than LM. Table l gives some differences between the two approaches. The detailed morphology revealed by EM may be called fine or submicroscopic structure/ultrastructure.
2 The direct comparison of LM and EM images of a structure requires that the magnifications be of the same order. Noting the magnification, on the 'scope or in the figure legend, allows one to adjust one's expectations of what may be seen, and should always be done.
3 A growing practice in histology and pathology is to fix and prepare the tissue by EM standards, imbed in plastic and cut semi-thin (l µm) sections for staining by modified LM methods. LM then reveals good cellular detail and fewer artefacts.
4 Two other techniques yield anatomical images - fibre-optic endoscopy and scanning EM, and are being digested by the anatomical texts. Endoscopy from its low magnification is marginal to histology, but related in that endoscopy is used to obtain biopsy specimens for histopathology.
SEM strengthens one's conception of microscopic structures, e.g., cilia, renal podocytes, bone under resorption, and effectively counters the unavoidable impression of structures existing only in two dimensions. (From hereon, EM is standard transmission electron microscopy.)
Table 2. Some differences between light and electron microscopy.
Light microscopy Electron microscopy -------------------------------------------------------------------------- Image is presented directly to the Image is in shades of green on eye. Image keeps the colours given the screen; photographically, the specimen by staining. only in black and white. Modest magnification to X 1500; High magnification, up to X 2,000,000 but a wider field of view and easier thus the range of magnification orientation is greater Resolving power to 0.25 µm. Resolving power to 1 nm (0.001µm.) Frozen sections can yield an image Processing of tissue takes a day at within 20 minutes. least. Crude techniques of preparation High resolution and magnification introduce many artefacts. demand good fixation (e.g. by (Histochemical methods are better.) vascular perfusion), cleanliness and careful cutting, adding up to fewer artefacts. Section thickness (1-30 µm) gives Very thin sections provide no a little depth for focus for depth of focus, but 3-D information appreciation of the third dimension. can be had from: (a) thicker sections Serial sections can be cut, viewed by high-voltage EM; (b) shadowed and used to build a composite image replicas of fractured surfaces; (c) or representation. scanning electron microscopy (SEM). Most materials and structures cannot Heavy metal staining gives a more be stained and viewed at the same comprehensive picture of membranes, time; stains are used selectively to granules, filaments, crystals, etc.; give a partial picture, e.g. a stain but this view is incomplete and even for mucus counterstained to show visible bodies can be improved by cell nuclei. varying the technique. Specimen can be large and Specimen is in vacuo. Its small size even alive. creates more problems with sampling and orientation. Light microscopy Electron microscopy